Cell-mediated killing by cytotoxic lymphocytes is an important immunologic defense against tumor cell proliferation, viral infection and transplanted tissue (Duke, R. C., et al. J. Exp. Med. 170:1451-1456 (1989)). Cytotoxic lymphocyte--mediated lysis is often associated with the formation of membrane lesions on target cells caused by exocytosis of cytoplasmic granules from cytolytic lymphocytes (Henkart, P. A., et al. J. Exp. Med. 160:75-93 (1984); Joag, S., et al. J. Cell Biochem. 39:239-252 (1989 )).
These granules contain proteoglycans (MacDermott, R. P., et al. J. Exp. Med. 162:1771-1787 (1985)) and several proteins (Tschopp, J., and Johngeneel, C. V. Biochemistry 27:2641-2646 (1988); Shinkai, Y., et al. Nature 334:525-527 (1988); Lichtenheld, M. G., et al. Nature 335:448-451 (1988); Tschopp, J., and Nabholz, M. Annu. Rev. Immunol. 8:279-302 (1990); Liu, C-C., et al. Cell 51:393-403 (1987); Tian, Q., et al. Cell 67:629-639 (1991)), including serine proteases (SP) (Tschopp, J., and Johngeneel, C. V. Biochemistry 27:2641 -2646 (1988)) and pore-forming protein (cytolysin) (PFP) (Shinkai, Y., et al. Nature 334:525-527 (1988); Lichtenheld, M. G., et al. Nature 335:448-451 (1988); Tschopp, J., and Nabholz, M. Annu. Rev. Immunol. 8:279-302 (1990)). PFP forms transmembrane ionic pores that produce membrane damage (Young, J. D. Physiol. Rev. 69:250-314 (1989); Ojcius, D. M., et al. Proc. Natl. Acad. Sci. USA 88:4621-4625 (1991)) but does not cause DNA fragmentation (Duke, R. C., et al. J. Exp. Med. 170:1451-1456 (1989)).
A direct role for SP in cytolysis has not been universally accepted (Henkart, P. A., et al. J. Immunol. 139:2398-2405 (1987); Berke, G. Immunol. Lett. 20:169-178 (1989); Ostergaard, H. L. and Clark, W. L. J. Immunol. 143:2120-2126 (1989)), despite the fact that a number of protease inhibitors can block cytotoxic T lymphocyte (CTL) --mediated lysis (Chang, T. W., and Eisen, J. E. Nature 124:1028-1033 (1980); Hudig, D., et al. J. Immunol. 147:1360-1368 (1991)). DNA fragmenting properties have been postulated for two members of the SP family (Munger, W. E., et al. Immunol. Rev. 103:99-109 (1988); Shi, L., et al. J. Exp. Med. 175:553-566 (1992)) when combined with PFP. Alternatively, it has been suggested that granules break down various extracellular matrix proteins (Simon, M. M., et al. Immunol. 60:219-230 (1987); Sayers, T. J., et al. J. Immunol. 148:292-300 (1992)) and may therefore be involved in the in vivo trafficking of NK cells and CTL or in modifying effector cell:target interactions.
Several serine protease genes from CTL and NK cells have been isolated and sequenced by several different laboratories (Zunino, et al. Biochem. Biophys. Acta. 967:331-340 (1988); Lobe, C. G., et al. Proc. Natl. Acad. Sci. USA 83:1448-1452 (1986); Lobe, C. G., et al. Science 232:858-861 (1986); Gershenfeld, H. K., and Weissman, I. L. Science 232:854-858 (1986); Jenne, D. E., and Tschopp, J. Immunol. Rev. 103:53-71 (1988); Brunet, J-F., et al. Nature 322:268-271 (1986); Kwon, B. S., et al. D-E. J. Exp. Med. 168:1839-1854 (1988); Schmid, J., and Weissman, C. J. Immunol. 139:250-256 (1987); Gershenfeld, H. K., et al. Proc. Nat'l Acad. Sci. USA 85:1184-1185 (1988); Manyak, C. L., et al. J. Immunol. 142:3703-3713 (1989); Zunino, S. J., et al. J. Immunol. 144:2001-2009 (1990)).
Some of these CDNAs encode the same protein, but seven different serine proteases have been identified. Seven serine proteases, granzymes A, B, C, D, E, F, and G, have been isolated and purified from murine granules (Maseon, D. and Tschopp, J. Cell 49:679-685 (1987)). Murine granzyme A is also known as Hanukah factor (Gershenfeld, H. K., and Weissman, I. L. Science 232:854-858 (1986)), TSP-1 (Simon, M. M., et al. Immunol. 60:219-230 (1987)), BLT-esterase (Pasternak, M. S., et al. Nature 314:743-745 (1985)), CTLA-3 (Brunet, J-F., et al. Nature 322:268-271 (1986)); and SE1 (Young, J. E.-E., et al. Cell 47:183-194 (1986)); murine granzyme B as CCP1 (Lobe, C. G., et al. Science 232:858-861 (1986)); and CTLA-1 (Brunet, J-F., et al. Nature 322:268-271 (1986)); granzyme C (Masson, D. and Tschopp, J. Cell 49:679-685 (1987)) as CCP2 (Lobe, C. G., et al. Science 232:858-861 (1986)); granzyme E as CCP3 (Bleackley, et al. FEBS Lett. 234:153-159 (1988)); and granzyme F as CCP4 (Bleackley, et al. FEBS Lett. 234:153-159 (1988)).
Granzymes A and B also have been isolated from human CTL granules and are homologous to the murine enzymes (Fruth, U., et al. Eur. J. Immunol. 17:1625-1633 (1987); Poe, M., et al. J. Biol. Chem. 266:98-103 (1991)). Human granzyme A is also known as HuTSP (Fruth, U., et al. Eur. J. Immunol. 17:1625-1633 (1987)), Hanukah factor (Gershenfeld, H. K., et al. Proc. Nat'l Acad. Sci. USA 85:1184-1185 (1988)), CTL tryptase (Poe, M., et al. J. Biol. Chem. 263 (26):13215-13222 (1988)), and granzyme 1 (Hameed, A., et al. J. Immunol. 141:3142-3147 (1988)); and human granzyme B as HLP (Schmid, J., and Weissman, C. J. Immunol. 139:250-256 (1987)), HSE26.1 (Trapani, J. A., et al. Proc. Nat'l. Acad. Sci. U.S.A. 85:6924-6928 (1988)), SECT (Caputo, A., et al. J. Biol. Chem. 263:6363-6369 (1988)), granzyme 2 (Hameed, A., et al. J. Immunol. 141:3142-3147 (1988)), and Q31 granzyme B (Poe, M., et al. J. Biol. Chem. 266:98-103 (1991)). Only one rat granzyme, RNKP-1, has been purified (Sayers, T. J., et al. J. Immunol. 148:292-300 (1992)) and sequenced (Zunino, S. J., et al. J. Immunol. 144:2001-2009 (1990)).
Thus far, the hydrolytic activities for only three enzymes, Tryptase, Chymase, and ASP-ase, encoded by the seven serine protease genes have been described. Granzymes A and F have been shown to exhibit tryptase activity (Jenne, D. E., and Tschopp, J. Immunol. Rev. 103:53-71 (1988); Jiang, S., et al. Protein Expression Purification 1:77-85 (1990)); granzyme B has shown predominantly ASP-ase activity and some Met-ase activity ( Odake, S., et al. Biochemistry 30:2217-2227 (1991); Poe, M., et al. J. Biol. Chem. 266:98-103 (1991)); and granzymes D, E, and F were predicted to have chymase activity (Odake, S., et al. Biochemistry 30:2217-2227 (1991)). Granzymes C and G, although tested, have not been shown to exhibit a specific enzymatic activity (Odake, S., et al. Biochemistry 30:2217-2227 (1991)). RNKP-1 has been shown to have ASP-ase activity.
With respect to the Met-ase activity exhibited by granzyme B, Odake, et al. suggested that this activity may belong to granzyme B because of the observation that the Met-ase and Asp-ase activities coelute from the Mono S column and the nearly identical inhibition rates of granzyme B with DCI when three different substrates are used. On the other hand, Odake, et al. stated that the observation by Poe, et al. (Poe, M., et al. J. Biol. Chem. 266:98-103 (1991)) that the Met-ase activity was separated from the Asp-ase activity during the purification of human granzyme B suggests the presence of different enzymes.
Other than granzyme B, no cytolytic granule proteins have been reported which exhibit Met-ase activity.
This invention describes the biochemical purification of a novel 30 kDa SP with Met-ase activity (RNK Met-1) from the cytolytic granules of the rat RNK-16 LGL leukemia. Isolation and sequencing of the cDNA encoding RNK Met-1 from a rat RNK-16 .lambda.gt-11 library reveals RNK Met-1 to be a unique SP gene with structural variations that distinguish it from other members of the SP family. The invention also describes the human serine protease and its cDNA clone obtained by screening the phage .lambda.gt-10 cDNA library from human Lopez LGL leukemia with the rat RNK-Met-1 cDNA clone.